Purpose: :
Prominin-1 (PROM1, AC133, CD133, PROML1) is a 5-transmembrane-domain glycoprotein located at plasma membrane protrusions and expressed in various tissues. To date, 5 different mutations in human PROM1 are known causing a variety of retinal degenerative disorders ranging from macular dystrophy via Stargardt disease, cone-rod dystrophy and bull’s eye maculopathy to retinitis pigmentosa. Recent results indicate that PROM1 plays a central role in photoreceptor outer segment disc morphogenesis. The PROM1 knock-out mouse displays disorganized outer segment disc structure and retinal degeneration. In this study we show a rescue of the knock-out phenotype by delivering wild-type PROM1 to the host photoreceptor cells.

Methods: :
Young, post-natal day 15 PROM1 knock-out mice (AC133-/-) were sub-retinally injected with rAAV vectors containing GFP or PROM1 transcripts. GFP was used for observation of target cells while PROM1 was used to rescue the knock-out phenotype. We tested rAAV serotype 5 and 2/8 vectors according to targeting specificity and transgene expression. PROM1 delivery was performed using rAAV2/8 vectors containing an ubiquitously active chicken beta-actin promoter driven transgene.

Results: :
We show that rAAV5 and rAAV2/8 have high photoreceptor targeting specificity and display a strong, injection-site restricted transgene expression. In few cases, single Müller-glia cells or second order neurons were targeted. PROM1 delivery by rAAV2/8 vectors to AC133-/- mice resulted in strong expression of PROM1 protein at its native sub-cellular location, i.e. the tip of the connecting cilium, 2 weeks after injection. Morphological differences between injected and non-injected eyes first occur 3 weeks after injection. We show a relative preservation of outer nuclear layer (ONL) thickness and photoreceptor morphology in treated eyes, in comparison to untreated controls, that show a strong decline in ONL thickness.

Conclusions: :
This study demonstrates a rescue of the PROM1 knock-out phenotype by delivering wild-type PROM1 to host photoreceptor cells using rAAV2/8 vector. Our results are in line with other studies showing promising results for gene therapeutical strategies for the treatment of retinal degenerative disorders and show the feasibility of gene therapeutical treatment of PROM1 mutations causing retinal degenerative diseases.